Glycosyltransferases for Human Milk Oligosaccharide Synthesis: From Structural Understanding to Engineering

Human milk oligosaccharides (HMOs) are vital for infant health by modulating gut microbiota and immune function. Due to challenges in natural extraction of HMOs, metabolic engineering using glycosyltransferases (GTs) offers a promising alternative. This review systematically explores GTs-mediated HMO biosynthesis, covering mechanisms, structural characterization, and protein engineering. It emphasizes recent advancements in the structural characterization and protein engineering of GTs, highlighting the strategies employed in each approach. Recent advances in structural analysis of four types of GTs (α-fucosyltransferases (FucTs), β-N-acetylglucosaminyltransferases (GlcNAcTs), β-galactosyltransferases (GalTs), and α-sialyltransferases (SiaTs)) have clarified their structure-function relationships, guiding optimization strategies like rational design, directed evolution, and domain truncation. Despite issues like low solubility and substrate specificity, tools like AlphaFold 3 have further guided enzyme optimization. Future efforts aim to expand GT diversity and improve microbial cell factories for scalable HMO production, meeting industrial and nutritional needs.